HPLC OF OLIGO(SIALIC ACIDS) - APPLICATION TO THE DETERMINATION OF THE MINIMAL CHAIN-LENGTH SERVING AS EXOGENOUS ACCEPTOR IN THE ENZYMATIC-SYNTHESIS OF COLOMINIC ACID

A rapid, sensitive and easy h.p.l.c. method was developed for the quantitative analysis of oligosialic acids. This procedure, which permits the complete separation (in 23 min) of several sialyloligomers with a degree of polymerization of between 1 and 16, has been employed to establish the minimal chain length of oligomer accepted, as an exogenous acceptor, by Escherichia coli K-235 sialyltransferase complex (ST) leading to the synthesis in vitro of colominic acid. We showed that this membrane-bound enzyme catalyses the direct transfer of Neu5Ac residues (one by one) from CMP-Neu5Ac to an exogenous acceptor molecule which contains at least three Neu5Ac residues. Free Neu5Ac or (Neu5Ac)2 were not recognized as substrates, whereas the maximal rate of polymer elongation was achieved when (Neu5Ac)5 was used as substrate.